Abstract
Background. Many children and adults with acute myeloid leukemia (AML) relapse or are incurable with current treatment modalities, highlighting a need for alternative therapies. Chimeric antigen receptor (CAR) T cells targeting CD123 have demonstrated potent anti-leukemia activity in murine xenograft models of human AML. However, CD123-redirected T cell treatment of mice engrafted with normal human hematopoietic cells resulted in profound myeloablation, raising concerns for hematologic toxicity in patients with AML who may be treated with such therapies. We hypothesized that T cell deletion after CD123-redirected T cell-induced eradication of AML could minimize this bystander toxicity without impairing leukemia control, thereby increasing the therapeutic window of anti-AML CAR T cell immunotherapy.
Methods. To test this hypothesis, we compared three termination strategies in human AML xenograft models: (1) T cell ablation with the anti-CD52 antibody alemtuzumab after treatment with T cells lentivirally-transduced with anti-CD123-41BB-CD3ζ (CART123), (2) T cell ablation with the anti-CD20 antibody rituximab after treatment with CART123 engineered to co-express CD20 (CART123/CD20), and (3) treatment with "biodegradable" anti-CD123 mRNA-electroporated CAR T cells (RNA-CART123). Mice engrafted with luciferase-expressing human AML cell lines (MOLM14, MOLM13, U937) or primary AML specimens (n=3) were treated with CD123-redirected CAR T cells as above. For T cell depletion studies, alemtuzumab 1 or 5 mg/kg was injected intraperitoneally (IP) at 1-4 weeks after 1x105-106 CART123 to determine optimal dosing and timing of T cell ablation. In subsequent studies, rituximab 10 mg/kg was injected IP 4 weeks after 1x105 -106 CART123/CD20, or 1x107 RNA-CART123 were injected intravenously at 5, 9, and 16 days after AML engraftment. Mice were followed by weekly bioluminescent imaging and/or quantitative flow cytometry analyses of blood, spleen, and/or bone marrow.
Results. CART123 treatment of CD123+ AML xenografts induced marked T cell expansion and leukemia eradication in vivo, resulting in long-term animal survival (p<0.0001 vs untransduced T cell-treated controls). Minimal xenogeneic graft-versus-host effects were observed. One dose of alemtuzumab rapidly eliminated T cells in all tested models with best efficacy of 5 mg/kg dosing at 4 weeks post-CART123. CART123/CD20 inhibited AML proliferation with similar kinetics to those of CART123, and 1 dose of rituximab at 4 weeks post-CART123/CD20 infusion rapidly eliminated T cells while preserving leukemia remission. Alemtuzumab or rituximab alone did not inhibit AML proliferation in non-CART123-treated xenograft models vs AML-only controls (p=1.00). Mice with CART123- or CART123/CD20-induced AML remission at time of T cell ablation remained leukemia-free for ≥12 weeks, and animal survival did not differ from that of CD123-redirected CAR T cell-treated mice that did not undergo T cell depletion (p=1.00). In contrast, early depletion of T cells with alemtuzumab at 1, 2, or 3 weeks post-CART123 resulted in failure to eliminate AML with subsequent leukemia progression and animal death. Furthermore, AML rechallenge of animals with alemtuzumab- or rituximab-ablated T cells resulted in rapid leukemia proliferation without T cell re-expansion, confirming completeness of T cell depletion. Non-ablated mice demonstrated CAR T cell re-expansion with rejection of CD123+ leukemia rechallenge (p<0.0001). RNA-CART123 rapidly eliminated AML and facilitated long-term animal survival, although RNA-CART123 had expectedly shorter persistence in vivo than did CART123 or CART123/CD20.
Conclusions. Alemtuzumab and rituximab completely eliminated CD123-redirected CAR T cells in human AML xenograft models. Sustained leukemia remission required CART123 or CART123/CD20 persistence for 4 weeks prior to T cell termination via alemtuzumab or rituximab post-CART123 or CART123/CD20, respectively. Ongoing studies are investigating efficacy of T cell elimination in additional xenograft models and against other anti-AML CAR T cell immunotherapies. Results from these studies may help to identify promising T cell termination strategies that will augment efficacy of CAR T cell therapy in patients with AML, particularly prior to stem cell transplantation. RNA-CART123 trials in patients with relapsed/refractory AML will open soon.
Disclosures
Off Label Use: depletion of CAR T cells with alemtuzumab or rituximab. Kenderian:Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Aplenc:Sigma Tau: Consultancy. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.
Pharmacokinetic and pharmacodynamic studies of CD19 CAR T cell in human leukaemic xenograft models with dual‐modality imaging